Noningestible separation barrier with plugged pores

ABSTRACT

A noningestible separation barrier having one or more pores of micropores (of one or more diameters) therein, with the pores or micropores being initially plugged with one or more materials selected for its/their solubility and/or integrity characteristics relative to certain environmental conditions. Ordinarily, the pores or micropores of the noningestible separation barrier are initially filled with at least one material having greater erodibility, under a given environmental condition, than the material constituting the separation barrier itself. The combination of the release rate (if any) or other membrane characteristic of the separation barrier, combined with the release rate and/or erosion life of the plugged pores, enables complex separations including variable release of cells, colloids, solutes or solvents over time, such as when the plugged pores remain intact until erosion is triggered by an environmental change such as solvent addition or ionic conditions or pH or thermal change. Particular applications include specialized receptacles and protocols for the preservation of rooster sperm and turkey sperm for use in commercial artificial insemination applications.

This application is a continuation of application Ser. No. 7/416,347,filed Oct. 6, 1989, which is a Continuation-in-Part of U.S. patentapplication Ser. No. 07/263,049, filed Oct. 26, 1988, abandoned.

FIELD OF THE INVENTION

The invention relates to separation technology, including membranologyand controlled release of solvents, solutes, or cells.

BACKGROUND OF THE INVENTION

Advances in the selective separation of matter have resulted in numerousdevelopments in a wide variety of industries. Attention first centeredon the science of membranology, beginning in about 1960, whenintegrally-skinned cellulose acetate hyperfiltration membranes weredeveloped for hyperfiltration desalination of salt water. Developmentsfollowed in the areas of hemodialysis, electrodialysis, reverse osmosis,ultrafiltration, cell harvesting, membrane bioreactors, microfiltration,gas separation, controlled time release, gel permeation chromatography,hollow fiber technology, non-cellulosic polymer membranes, ionomermembranes, copolymer membranes, crosslinkable thermoplastic polymermembranes, emulsion-type liquid membranes and others. These innovationshave gained general acceptance, and separation materials form the abovedisciplines are in widespread use in medical processes, pharmaceuticalresearch and production, industrial processes, research tools andconsumer products including consumer products packaging materials.

Controlled release of pharmaceuticals is now possible due to varioustechnologies, which include application of slow-dissolving coatings tooral dosage form drugs. U.S. Pat. No. 4,755,180 discloses an oral drugdosage form in which an erodible material, formed as a film around thedrug during manufacture, is eroded or leached from the wall of thedosage form, such erosion or leaching enabling controlled release ofpharmaceutically active agents to the gastrointestinal environment. Theerodible materials disclosed in U.S. Pat. No. 4,755,180 are typical ofthe polysaccharide (sugar) coatings common in such applications:poly(glycolic) or poly(lactic) acid compositions, gelatinouscompositions, or leachable polysaccharides, salts or oxides. Entericcoatings are also known in the art, which do not dissolve in the stomachbut allow enteric delivery of an orally dosed drug.

Although means are known for moving ions or molecules--or solvents--atsimple rates, such as the "zero-order" or "first-order" release kineticstypical in controlled release pharmaceuticals, no technology hasheretofore provided a noningestible means for complex separationtechnology in which the separation kinetics may change over time, inresponse to an environmental stimulus, in a pre-planned orpre-programmed manner; moving components into and out of a container toprovide a better environment for the retained materials; or release ofcells in a controlled manner. Accordingly, a need remains for aseparation barrier which can provide complex separation protocols forparticular separation applications entirely different from the oralpharmaceutical dosage forms.

SUMMARY OF THE INVENTION

In order to meet this need, the present invention is a noningestibleseparation barrier having one or more pores or micropores of one or moresizes therein, with the pores or micropores being initially plugged witha material selected for its solubility and/or integrity characteristicsrelative to certain environmental conditions. Ordinarily, the pores ormicropores of the separation barrier are initially filled with at leastone material having greater erodibility, under a given environmentalcondition, than the material constituting the separation barrier itself.Ordinarily, the separation barrier with its plugged pores is preparedprior to filling with the materials to be separated. The combination ofthe release rate (if any) or other membrane characteristic of theseparation barrier, combined with the release rate and/or erosion lifeof the plugged pores, enables complex separations including variablerelease of cells, colloids, solutes or solvents over time, such as whenthe plugged pores remain intact until erosion is triggered by anenvironmental change such as solvent addition, pH, or thermal orradiation (ultraviolet light, etc.) change. Particular applicationsinclude specialized protocols for the preservation of rooster sperm andturkey sperm for use in commercial artificial insemination applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a hollow tubule (hollow fiber) separation barrier havingplugged micropores;

FIG. 2 is a side elevational view of a separation barrier membranehaving small and large pores plugged with two different polymers,respectively;

FIG. 3 illustrates a three-dimensional rectangular construct, closed onfive sides, having plugged micropores on each closed polymer facethereof, with the pores in each closed polymer face being plugged withmaterials having different erosion characteristics;

FIG. 4 is a schematic illustration (plan view) of a chamber whichcontains exchange medium and into which are placed six containersaccording to the present invention; and

FIG. 5 is a section taken along lines V--V of FIG. 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a noningestible separation barrier having oneor more pores or micropores of one or more sizes therein, plugged with amaterial selected for its solubility or erodibility upon exposure tocertain environmental conditions. The material forming the separationbarrier itself may also have erodibility or solubility, as long as theseparation barrier and plugged pores do not consist of the identicalcomposition of matter. The combination of the release rate (if any) orother membrane characteristic of the separation barrier, combined withthe release rate and/or erosion life or lives of the plugged pores,enables complex separations including variable release of cells,colloids, solutes or solvents over time.

Separation barriers having plugged pores, according to the presentinvention, have a wide variety of uses in a myriad of industries.Suitable applications include cell cultures and cryobiology,preingestion preservation and storage of food and pharmaceuticals,shelf-life extension of polymers, proteins and other products, and thecontaining, transporting and dispensing of active agents includingcells, herbicides, pesticides, fertilizers, disinfectants, indoor airfresheners, and cell growth nutrients or other biologically activeagents for use in laboratory or industrial settings.

Because the scope of the present invention is particularly broad, inview of the numerous variations in the product and process claimed aswell as widespread applications thereof, a specific example is the bestintroductory illustration of the present separation barrier havinginitially plugged pores. For example, cryopreservaton of spermatozoa forartificial insemination, in certain species, has heretofore requiredprocess steps which can be eliminated by the complex separationprotocols possible with the present invention. Ordinarily,cryopreservation of spermatozoa requires attention to threeconsiderations. First, cryopreservation requires selection of anappropriate container, including appropriate volume, dimensions,material, thermal properties, etc., with the appropriate choices usuallyincluding nonporous glass ampules or vials, plastic vials or straws ormetal tubes. Second, a cryoprotectant must be selected to ensuresurvival of the cells during cooling from 38° C. to 0°-5° C., subsequentcooling to minus 196° C., and subsequent rewarming above 0° C.Conventional cryoprotectants include egg yolk, lipoproteins, milkproteins, glycerol, dimethylsulfoxide, polyethylene glycol, sugar andothers. Third, controlled post-thaw modification of the cellular andintracellular environment is essential for certain species. For example,rooster sperm must be serially diluted with a medium free from thecryoprotectant, followed by centrifugation and resuspension in the samecryoprotectant-free medium. This latter process, although cumbersome,has in the past been essential to avoid the inevitable contraceptiveeffect of the presence of the cryoprotectant around and within the spermat the moment of artificial insemination.

As is explained in greater detail in Example 1, below, the presentseparation barrier having plugged pores simplifies thawing and removalof the cryoprotectant from the spermatozoa of the rooster. Referring nowto FIG. 1, the terminal portion of a sealed polymeric tubule isillustrated, comprising the tubule 10 having plugged micropores 12therein and an end seal 14. The end of the tubule not shown may comprisean open end; alternately, the seal 14 may be removed or originallyomitted to leave an open end to the tubule 10. The tubule may beprelabelled for identification or may be adapted for insertion of alabelled plug in the open end. Collected rooster semen, evaluated,pooled, extended with media and processed by means known in the art, iscooled to 5° C., mixed with cryoprotectant (if cryoprotectant is absentfrom the original extension media), and charged to the interior of thetubule 10 of FIG. 1. The tubule 10, a polymeric barrier havingpolymer-plugged micropores 12, constitutes the storage container forcryopreservation of the rooster semen. The open end of the tubule isthen sealed. The sealed tubule and its contents are then cooled at oneor more controlled rates, as known in the art, to -196° C.

Prior to insemination of a group of chickens, a tubule 10 is transferredfrom cryopreservation storage at minus 196° C. (liquid nitrogen) into athawing solution known in the art, and after initial thawing the tubule10 is subsequently transferred to a post-thaw treatment solution ofappropriate temperature, composition and oxygenation. In the post-thawtreatment solution, the action of at least one of the constituents ofthe post-thaw treatment solution on the plugged micropores 12 opens themicropores 12 and allows controlled egress of the cryoprotectant fromthe environment around and within the rooster sperm and simultaneouslypermits controlled ingress of the post-thaw treatment solution into thetubule 10. The micropores 12 are, of course, smaller than the spermcells. After a 10-60 minute treatment period, sperm in the tubule areready for use. The tubule 10 may be used, along with appropriateauxiliary mechanical devices, to disperse the sperm directly into one ormore hens for artificial insemination of the chicken flock, or the spermmay be transferred from the tube into an appropriate auxiliarymechanical device for insemination. As an illustration of the concept ofthe present invention, the tubule 10 is insoluble in the liquid nitrogenat minus 196° C., as are the polymer plugged micropores 12. Both thetubule 10 and plugged micropores 12 are likewise insoluble or notcompletely eroded in t he thawing solutions known in the art. Thus,during two periods of use, the tubule 10 undergoes no separation orchange in separation kinetics with respect to its contents or structure.However, upon contact with the post-thaw treatment solution, the actionof at least one constituent of the post-thaw treatment solutionconstitutes the environmental factor which commences or completesdissolution of a selected polymer within the plugged micropores 12, sothat pre-determined time- and rate-controlled egress of cryoprotectantand ingress of medium is accomplished. If two or more cryoprotectantsare used, they can be removed selectively through pores having differentsizes and/or erosion characteristics (as suggested in FIGS. 2 and 3).

It will be understood by those skilled in the art that both the pluggedmicropores 12 and the tubule 10 (the separation barrier itself) may bothbe fabricated of materials soluble or erodible under certain conditions.For example, the plugged micropores 12 may be moderately soluble inhuman plasma, with the tubule 10 being only somewhat soluble in humanplasma, so that an entire structure comprising a small tubule 10according to FIG. 1, filled with a pharmaceutically active agent, wouldconstitute a human subcutaneous controlled release drug implant whichwould not release the agent for some period of time, then release theagent at a controlled rate, and ultimately completely erode. Likewise,tubules 10 according to FIG. 1, or other structures such as that shownin FIG. 3, may be used in the cryopreservation of a wide variety ofcells and cell cultures, with the polymer or other material for pluggingthe plugged micropore 12 being selected in reliance on the reaction orrelease kinetics required by the particular post-thawing parametersrequired for the preserved cells. Additional applications includecontrolled release of nutrients to cells or organisms; bacteria;herbicides; and release of alkaline agents to neutralize water or soil,each triggered by the appropriate change in environmental conditions.

Separation barriers according to the present invention may be fabricatedfrom a wide variety of materials including polymers, ceramics, metalsand natural and semi-synthetic cellulosics. More particularly, theseparation barrier may consist of a wide variety of materials andpolymers including polyether compositions, polyethylene andpolypropylene polymers, polyvinyl polymers, moisture vapor permeableurethanes and other polyurethanes, polycarbonate polymers, cellulosics,semi-synthetic cellulosics, ceramics, metals, natural resins includingrubbers, etc. When appropriate, the separation barrier may be fabricatedof one or more of the compositions also suitable for use in plugging thepores or micropores in the separation barrier, which compositionsinclude cellulosics (i.e., hydroxypropylmethylcellulose),polyelectrolyte complexes, polysulfone compositions, acrylic polymers,cellulose acetates, Dynel® compositions, polyacrylonitrile compositions,polyvinylpyrrolidone polymers, cellulosic composites on polyvinylchloride, and other materials known in the membranologic arts. Alsofeasible are selectively soluble salt or polysaccharide crystals as longas the dissolution of same does not poison adjacent biologic media andis not toxic to cells in or near the barrier. Selection of one or morematerials for the preparation of the separation barrier having pluggedpores will be dictated directly by the specific application intended andthe release kinetics desired. Also, the separation barrier may beconstructed for one-time use, or may be designed for multiple uses i.e.,when the barrier device is returned to the producer for replugging andrefilling.

As an overview of the various applications of the different types of"plugger" polymers, water-soluble materials included within the pores ormicropores of the separation barrier will have applicability at least tocryobiologic preservation of spermatozoa (poultry, human, horse, fish,pig, sheep, dog, mouse, rat, etc.); oocytes (cattle, human, horse, pig,mouse, rat, etc.); embryos (human, cattle horse, mice, dogs, etc.) ofall species of mammals with physiological need and economic advantage;human pancreas B-cells; human corneas; primary cell cultures; and seeds.Controlled release of animal agricultural hormones; plant agriculturalfertilizers, herbicides, and pesticides; seeds; agriculture nutrients,pharmaceuticals and larvae; and home insecticides, fertilizers andherbicides is also envisioned. Thermal sensitive polymer plugs haveapplication in frost damage control: erodibility of the plugs, in theseparation barrier pores, can upon decreasing temperature releasefrost-protective bacteria onto crops, fields or orchards. Polymer plugshaving pH sensitivity may be applied to the non-oral controlled releaseof pharmaceuticals to animals and humans in pH-fluctuating anatomicareas, and likewise have application in the ecosystem to deliver theappropriate acid or base upon a drop or increase in environmental pHconditions. Photo-inducible polymer erodibility can release agriculturalmaterials after sufficient exposure to sunlight. Other environmentalconditions including electricity, sound and magnetism can also beharnessed by appropriate "plugger" material types, which erode at theappropriate cued time. Finally, environmental cues can triggerpolymerization, instead of erosion, of the material in the pores ormicropores. Other examples will be readily evident to those skilled inthe art.

Referring now to FIG. 2, a side elevational view of a membrane 20 isshown having pores of two different diameters. The large pores 22 arefilled with a first polymer composition and the small pores 24 arefilled with a second and different polymer composition. By appropriateselection of the material constituting the membrane 20 and the materialwithin each of the large pores 22 and the small pores 24, any one ofthree or all three separation kinetics attributable to the respectivepolymers may be controlled by appropriate changes in the corollaryenvironmental conditions including temperature, pH, presence of specificsolvents, electrical charge, magnetic field, light waves, or soundenergy. For example, if the largest pores open last, the largestseparable particles cordoned by the membrane will diffuse last. Themembrane 20 of FIG. 2 may be used in lieu of other prior art separationmembranes, when the sequential or selective separation kinetics possiblewith the membrane 20 are desired or necessary for a given application.Such a membrane can be directly fabricated into a variety of shapes, orused as a sheet for later insertion into an appropriate container.

FIG. 3 illustrates a three-dimensional rectangular construct having fiveclosed sides, or faces. The rectangular construct 30 is fabricated of apolymer, and the largest closed face thereof has polymer-plugged pores32 therein. The remaining closed faces shown have polymer-plugged pores34 and 36 therein. Polymer plugged pores 32, 34, and 36 are filled witha second, third, and fourth polymer, respectively. As with the deviceshown in FIG. 2, the rectangular construct 30 functions asbuilding-block type unit, and can be used alone or in an array of anumber of identical rectangular constructs 30 for laboratory orproduction use in a variety of configurations. Complex sequential orselective separations are possible due to the varied polymer plugs inthe polymer-plugged pores 32, 34, and 36. The remaining closed faces notshown may also contain plugged pores. The rectangular construct 30 isrepresentative of a commercial embodiment of the present separationbarrier having plugged pores, in which units may be assembled by theuser as needed.

Referring once again to the present separation barrier generally, theplugged pores may be true plugged pores, i.e., the pores in theseparation barrier may be filled to the extent of the pore cavity only,or the plugged pores may be plugged by means of a continuous coating onone or both sides of the separation barrier to plug the pores by coatingover them. Also, with respect to either the separation barrier itself orthe pore-plugging material, or both, additional treating materials maybe used to alter the chemical and physical properties of the polymers,such as for altering toxicity, adapting polar and non-polar materialsfor their specific intended applications, or increasing bondingproperties.

Although the structures represented in FIGS. 1-3 are illustrative,numerous other containers may be fashioned in view of the concept of theinvention. A container need not be homogeneous, for example, but couldhave sides each of different materials. A six-sided construct couldinclude six different materials in the different-sized pores of eachrespective side, for example, which construct would then effect uniqueseparation kinetics by means of each of its separate sides.

Dimensions for the tubule of FIG. 1, for cryopreservation of sperm,range from 1-80 mm. in diameter or more, with 1-5 mm. diameters beingpreferred. The tubules may range in length from about 5 mm. to 10centimeters or more, with about 50-200 mm. being preferred. Pores ormicropores may be of any size. For many separations, pores of 0.2 to 0.6micrometers effective diameter are preferred, but for some applicationsonly pores greater than 0.6 micrometers are appropriate. Often pores canbe larger than the pores in prior art separation membranes due to thenature of the materials to be retained and the initial pluggedconfiguration of the pores. Other embodiments of the invention may havewidely varied dimensions depending upon the intended application.

Polymers which can provide a persistent plug in a number of environmentsinclude ethyl cellulose, among others. For applications requiring watererodible plugs, methyl cellulose plugs are appropriate. The use ofpolyvinylpyrrolidone enables thermal transition opening of the porescontaining it, and carboxymethylcellulose allows pores to open inresponse to pH shift. Detergent activated, photoactivatable, and othercompositions known in the art can be selected as needed.

A further particular application of the present invention is in theproduction of fertilized turkey eggs, which eggs can be producedcommercially only by artificial insemination (AI). Prior art methodspermit commercial holding of turkey sperm for only six to eight hoursafter collection, which allows only about two hours for transport of theturkey sperm. In the laboratory, oxygenation of turkey sperm hasincreasing the holding time to about 24 hours, but the laboratorytechniques have been impractical on a commercial basis. By applying theproducts and methods according to the present invention, turkey spermviability and quality should be maintained for up to 48 hours.Preservation of turkey sperm is discussed further below and in ExampleIV herewith.

The present invention provides for the use of unique containers andstorage conditions for preservation of turkey sperm. The container isuniquely constructed with pores that are ordinarily initiallyimpermeable (allowing easy loading and handling) but which open rapidlyafter the container of turkey semen is placed in contact with exchangesolutions. Precise control of temperature and oxygen content andcontinuous exchange of nutrients and antioxidants into the spermsuspension, without damage to the sperm, is possible using the containeralong with an optional microprocessor controlled system. This will allowthe industry to create "super stud farms" with the potential for totalreorganization of the distribution of germ plasm.

Despite current limitations of semen holding/storage, the industryexists only because of use of AI for reproduction. Passage from thebreeder to the commercial producers is by sale of eggs of the individualmale and female lines as needed for the construction of the commercialgenetic product. If held or stored semen were available, industry needscould be better satisfied and several problems would be eliminated.First, geographical separation of males and females is desirable toallow optimum management of each. Increasing the holding time makespossible wider use of stud farms. Second, coordinating purchase of maleline with female eggs to meet future needs is difficult. Shortages ofone line are costly. Held semen would alleviate this problem. Third,selection for production traits of individual males could be increased,thus decreasing the cost per pound of meat to the consumer. Fourth,purchase of semen instead of eggs removes the cost and nuisance ofoffsex disposal. Fifth, the breeder will have greater control over thequality of the commercial meat bird, again to the benefit of theconsumer. Finally, the breeder will have greater protection of the maleline germ plasm.

The general approach to preservation of turkey sperm is as follows.Turkey toms (e.g., male line meat strain) are ordinarily housed in aroom with 14:10 hour light:dark cycle. Semen is collected by massageevery 3-4 days from a group of toms and pooled within a few minutes.Sperm quality is determined by several tests, as are known to the art.Several extenders have been developed for holding turkey sperm and 5° C.and are known in the art, and these extenders may be admixed withacceptable turkey semen and placed within plugged-porous containersaccording to the present invention.

The appropriate plugged porous container is ordinarily fabricated fromnylon "frames" and specially prepared polysulfone membranes with 0.22 μmpores, with the pores initially sealed using methyl cellulose and ethylcellulose. Identical membranes are fastened to both large faces of thecontainer using IV-activated glue. These containers are nontoxic toturkey sperm and have pores that open in a controlled and repeatablemanner. Transmembrane flux of small molecules (<10,000 kD) in theinternal or external diluent has a half-time of about 2-5 min.; spermcannot pass through the membrane. The above-identified polymers areexemplary.

The containers are incubated in special environmental control units.Chambers are fabricated and fitted with circulation pumps, miniatureoxygen sensors, small air pumps, electronically controlled air nitrogenvalves, and a small oxygenation reservoir. The total volume of thechamber is about 30 ml; the chamber is preferably fitted with controlcircuits. Alternatively, the desired oxygen content in the medium can beachieved using appropriately blended gas mixtures and a constant rate ofaddition with a diffuser, as are known in the art. When the containers(containing suitable diluents and extenders along with the turkey semen)are cooled and held within the appropriate exchange solutions in thecontrolled chamber, turkey sperm viability and quality can be maintainedfor >18, and possibly >48, hours.

Although the use of a porous container held within a chamber containingdialyzing fluid is novel, the appropriate extenders, diluents andexchange solutions are known in the art. The environmental controlchamber regulates oxygen tension and temperature of turkey sperm towhich have been added diluents and/or extenders; the present inventionprovides the heretofore unknown means by which cooling, selectiveoxygenation, antioxidant treatment, etc., of turkey sperm may be scaledup to a commercially feasible level using porous containers. Aparticularly noteworthy aspect of the cooling/selective oxygenationpreservation of turkey sperm is the option whereby extended and/ordiluted turkey semen may be charged to a porous container in which thepores are not initially plugged, simultaneous with immersion of theporous container in the exchange solution. (The pores have a diametersmaller than the diameter of the turkey sperm cells, of course.)

Referring now to FIGS. 4 and 5, an apparatus suitable for preservationof turkey semen is illustrated schematically in plan and sectionalviews, respectively. A five-walled chamber 40 has control means forcontrolling temperature and oxygenation (for example, oxygenconcentration can be monitored with a miniature oxygen sensor connectedto a multiplex A/D converter, with optional electronic data storage) andacts as a bath-type receptacle for turkey semen receiving containers 42.Exchange fluid 44 selectively enters and exits the chamber 40 via inlet46 and outlet 48 as desired, and the exchange fluid 44 can enter andexit the containers 42 depending upon the constituents of the fluid andthe pore size of the container(s).

The invention will be more particularly described by means of thefollowing illustrative examples.

EXAMPLE I

Frozen rooster semen is not successfully used commercially because thecryoprotectant most effective for spermatozoa survival during freezingand thawing, glycerol, also demonstrates contraceptive properties afterartificial insemination. By prior art methods this cryoprotectant isremoved by serial dilution followed by centrifugation or conventionaldialysis. Such processes successfully reduce cryoprotectantconcentration but are commercially impractical.

Because of the contraceptive effect of the glycerol, therefore, theglycerol must be removed from the avian spermatozoa prior to artificialinsemination. However, rapid removal of glycerol from avian spermatozoadamages the spermatozoa cells, probably as a consequence of rapidmovement of glycerol across the cell membrane, which alters thecharacteristics of the membrane and the viability of the cell. Slowremoval of glycerol from cryopreserved rooster semen is therefore anecessity, notwithstanding the typical high cost of achieving itaccording to prior art techniques.

Use of the present separation barrier having plugged pores, for thecontrolled removal of cryoprotectant from rooster spermatozoa, isdescribed below.

A group of roosters raised by an integrated breeder is selected, whichroosters possess a virtually identical genetic background and thereforetransmit the same phenotype. Semen is collected from this group ofroosters, a collection unit, with the semen then being evaluated,pooled, extended, and cooled by means known in the art. (Oxygenation isrequired if the cells are held above 15° C. because avian spermatozoaare highly dependent on oxidative metabolism unless they are cooled tonear 5° C. or frozen.) Initial cooling proceeds to 5° C. After theextended semen achieves 5° C., glycerol is added as a cryoprotectant (ifnot present in the original extender), and the cryoprotected extendedsemen then is immediately charged to hollow tubules having pluggedmicropores such as the tubule illustrated in FIG. 1.

More particularly, the hollow tubule is a cylindrical polymericseparation barrier open at one end and having plugged pores therein,having the following features: (1) impermeable for 20-30 minutes toaqueous solutions placed inside at 5° C.; 2) impermeable to viruses orbacteria coming in contact with the outside of the tubule, while thepores remain plugged; (3) resistant to immersion and long-term storagein liquid nitrogen; (4) resistant for 6-300 seconds to warming inaqueous solutions at 2°-75° C.; (5) has pores partially soluble upon5-20 minutes' further immersion in aqueous post-thaw treatment solutionsat 5°-38° C.; and 6) develops pores of a size which permit onlymolecules below a certain size to pass. These features are accomplishedby means of a microporous hollow fiber having the followingcharacteristics; (1) wall thickness, 0.5 mm; length, 60 mm.; anddiameter, 4 mm. (other possible configurations include: a right cylinder(30-40 mm. diameter and 5-10 mm. in length) of a nonporous material,such as polystyrene, to which is attached a "lid" of the microporousmembrane described herein, or a flattened tube 2 mm.×20 mm.×60 mm.); (2)separation barrier constructed of polyethylene (or other acceptablepolymer); and (3) 0.2 micrometer micropores plugged with methylcellulose.

If a three-dimensional rectangular device is used, to provide greatercapacity or an alternative packaging process, it would have fiveimpermeable sides, fabricated from polystyrene or another material,forming a square or rectangular shape (25 mm,×25-40 mm.×5-10 mm.) andthe sixth surface would be a microporous membrane with plugged pores,applied and sealed as a lid.

Cryopreservation of the semen in the hollow tubules (or tubes or othercontainers described above) is effected by means known in the art.Shortly before insemination of a flock of chickens, a tubule is removedfrom liquid nitrogen in storage at minus 196° C. and is transferred to athawing solution. After thawing, the hollow tubule is transferred to anaqueous post-thaw treatment solution including additives necessary forthe optimal survival of the avian spermatozoa (with oxygenation, ifappropriate). Upon immersion in the post-thaw treatment solution, theplugged pores open to permit controlled and relatively slow egress ofglycerol cryoprotectant from the solution within the container, andhence from the spermatozoa, and simultaneous controlled ingress of theexternal medium to surround the thawed rooster sperm. The thawed andtreated rooster sperm then are ready for use in artificial insemination.

EXAMPLE II

A second example is provided to illustrate the concept of two differentrates of release, as well as an extended time for initiation of poreopening.

For human sperm, both glycerol and crude lipid micelles (e.g., egg yolklipoprotein particles) are used in the art of cryopreservation. It isdesirable, after thawing, to remove the glycerol slowly (as for roostersperm) and then rapidly remove the egg yolk lipoprotein particles. Toaccomplish this objective, the general process of Example I is followedexcept that the initial extender contains egg yolk and is centrifuged(20,000×g for 30 minutes, for example) or filtered (through a 0.2 or 0.4micrometer filter) to remove material larger than the dimension desiredfor the colloidal particles, and glycerol is added after the extendedsperm are cooled to 5° C.

The container is a three dimensional rectangular construct (although theshape is not important) with four nonporous sides. One of the othersides is a microporous membrane with pores (about 0.2 micrometersdiameter) plugged with a material (i.e., high ratio of methyl celluloseto ethyl cellulose) having erodibility characteristics suitable for thecontrolled removal of glycerol. These pores open soon after immersion ofthe container into the thawing solution. The last face consists of amicroporous membrane with different size pores (0.3-0.5 micrometers)plugged with a different material (i.e., low ratio of methyl celluloseto ethyl cellulose) that is rapidly eroded after a time delay (30-40minutes) to allow rapid egress of the egg yolk lipoproteins and furtherentrance of the treatment medium.

EXAMPLE III

A third example is provided to illustrate release of soluble materialsto the environment and an alternative mode of release in response tochanging environmental conditions.

Nutrients may be provided to cultured cells held within "bioreactors".Continuous culture of cells for purpose of production of pharmaceuticalsand other bioproducts requires (at a minimum): removal of the desiredproducts as they are excreted into the growth medium; removal of toxicside products from the medium; and selective means for replenishingnutrients and regulatory substances necessary for continued culture ofthe cells.

Controlled release of nutrients and regulatory substances is achieved bythe following process. A hollow tube, composed of one or more poresplugged with one or more materials chosen to allow selective openingunder environmental stimulus, is filled with a sterile suspension (solidor liquid) or solution of the materials needed for continuous growth andproduction by the specific cells under culture. Examples includevitamins, hormones, trace metals, etc. The surface of the tube then issterilized, yielding a container that allows long thermal storage of thematerials to be added to the culture, and facile insertion into thebioreactor network as needed.

The tube is inserted into the culture (directly or into circulatingmedia) as needed. In response to this new environment, the pores open torelease the materials to the cells. The environmental signal is either apassive (water based erosion of the pore plug, as outlined for thetreatment of rooster sperm) or an active response to the changingculture conditions (pH shift as induced by production of acidby-products (e.g., lactic acid) by the cells). Such a container can alsobe used in a large scale agriculture environment, i.e., addition ofnutrients for growth of fish on a commercial scale.

Note: We have never done all the parts of this example.

EXAMPLE IV

A plastic chamber holds six rectangular plugged-pore containers plugexchange solutions. The chamber is fitted with control circuits, an"oxystat," a control means which enables maintenance of constant O₂tension by way of differential addition of air or N₂, and with meansknown in the art for temperature control, pumping and valving.

Turkey semen is collected, pooled and extended by means known in theart; the extended turkey sperm is cooled at a linear rate totemperatures between 5° C. and 20° C. and ≧1.5 ml aliquots are chargedto each of the six rectangular plugged-pore containers. The containershave inside dimensions of 3.5×17×36 millimeters, although containerhaving inside diameters of 3.0×17×22 millimeters could be substituted.The two largest walls of the containers are constructed of polysulfonemembranes having 0.22 micrometer pores, with the pores initially sealedwith methyl cellulose and ethyl cellulose. The containers are placed inthe chamber containing exchange fluid at the desired temperature (5°-20°C.); the pores of the containers are no longer plugged after severalminutes. Cooling and dialysis can continue for the holding period.

Although the invention has been described particularly above, theinvention is only to be limited insofar as is set forth in theaccompanying claims.

We claim:
 1. A method for separating a mixture including spermatozoa andglycerol comprising:(a) selecting a noningestible barrier materialsuitable for containing said mixture; (b) providing at least one pore tosaid barrier material, which pore is small enough to prevent passage ofat least one of the said spermatozoa or glycerol; (c) plugging saidpore, with a material responsive to an environmental stimulus, to yielda plugged pore and a plugged barrier; (d) isolating together saidmixture by means of said plugged barrier; and (e) exposing said pluggedbarrier at a preselected time to the environmental stimulus to whichsaid plug in said pore is responsive.
 2. A method for separatingglycerol from frozen rooster sperm, comprising:(a) selecting acylindrical polymeric separation barrier open at one end and havingplugged pores therein, wherein said plugged pores incorporate a methylcellulose composition, wherein said plugged pores are impermeable for atleast 20 minutes to aqueous solutions at 5° C., wherein said pluggedpores are impermeable to viruses and resistant to long-term storage inliquid nitrogen, wherein said plugged pores are further resistant towarming in aqueous solutions and temperatures of 2°-75° C. for 6-300seconds, and wherein said plugged pores are at least partially solublein one or more aqueous post-thaw treatment solutions at a temperature ofbetween about 5°-38° C., and further wherein said cylindrical polymericseparation barrier has a wall thickness of about 0.5 millimeters, alength of about 60 millimeters and a diameter of about 4 millimeters,and further wherein said cylindrical polymeric separation barrier isconstructed of polyethylene; (b) collecting rooster semen andevaluating, pooling, extending and cooling it; (c) charging theevaluated, pooled, extended and cooled rooster semen to said cylindricalpolymeric separation barrier, sealing the open end, and storing saidcylindrical polymeric separation barrier in liquid nitrogen at -196° C.;(d) thawing aid cylindrical polymeric separation barrier in a thawingsolution; (e) transferring said cylindrical polymeric separation barrierfrom said thawing solution to an aqueous post-thaw treatment solution;(f) maintaining said cylindrical polymeric separation barrier in saidaqueous post-thaw treatment solution for an amount of time effective topermit controlled and relatively slow egress of glycerol cryoprotectantfrom within said cylindrical polymeric separation barrier, withsimultaneous controlled ingress of the aqueous post-thaw treatmentsolution into the thawed rooster sperm; and (g) using the rooster spermas prepared in steps (a) through (f) above in the artificialinsemination of chickens.
 3. A method for preserving turkey semen,comprising:(a) collecting turkey semen, and adding an optional diluentor optional extender; (b) charging a quantity of said turkey semen intoa receptacle having at least one plugged pore therein and further havingan access means thereto other than said plugged pore; and (c)controlling the temperature and oxygenation of said turkey sementhroughout a period of storage of said turkey semen.
 4. The methodaccording to claim 3, wherein step (b) further comprises the step of:(b)charging a quantity of said turkey semen into a receptacle having atleast one pore therein, said pore being plugged at the time of thecharging of the turkey semen with an erodible composition;
 5. The methodaccording to claim 3, wherein step (b) further comprises the step of:(b)charging a quantity of said turkey semen into a receptacle having atleast one micropore therein, said micropore being plugged at the time ofthe charging of the turkey semen with one or more cellulosiccompositions.
 6. The method according to claim 5, wherein said microporehas a diameter of 0.22 micrometers.
 7. The method according to claim 5,wherein step (c) further comprises the step of:(c) controlling thetemperature and oxygenation of said turkey semen by bathing saidreceptacle in a chamber equipped with control means therefor andcontaining exchange fluid therein.
 8. The method according to claim 7,wherein each of said receptacle and said chamber is rectangular.
 9. Amethod for preserving and separating a mixture of two or more materialsincluding semen and an optionally added diluent or extendercomprising:(a) collecting semen and adding an optional diluent or anoptional extender thereto to form said mixture; (b) selecting areceptacle having at least one plugged pore therein and further havingan access means thereto other than said plugged pore; (c) charging aquantity of said mixture into said receptacle; (d) controlling thetemperature of the mixture throughout a period of storage of saidmixture; (e) removing said receptacle from the temperature controlledstorage; (f) transferring said receptacle to a post-storage treatmentsolution; and (g) maintaining said receptacle in said treatment solutionfor an amount of time effective to permit controlled and relatively slowegress of said optional diluent or extender from within said receptacle,with simultaneous controlled ingress of the post-storage treatmentsolution into the semen.